scholarly journals In Vitro ELISA and Cell-Based Assays Confirm the Low Immunogenicity of VNAR Therapeutic Constructs in a Mouse Model of Human RA: An Encouraging Milestone to Further Clinical Drug Development

2020 ◽  
Vol 2020 ◽  
pp. 1-14
Author(s):  
Obinna C. Ubah ◽  
Andrew J. Porter ◽  
Caroline J. Barelle
Keyword(s):  
Mouse Model ◽  
Solid Phase ◽  
Molecular Architecture ◽  
Serum Samples ◽  
Complete Control ◽  
Drug Levels ◽  
Trough Serum ◽  
The Impact

Anti-drug antibodies (ADAs), specific for biotherapeutic drugs, are associated with reduced serum drug levels and compromised therapeutic response. The impact of ADA on the bioavailability and clinical efficacy of blockbuster anti-hTNF-α monoclonal antibodies is well recognised, especially for adalimumab and infliximab treatments, with the large and complex molecular architecture of classical immunoglobulin antibody drugs, in part, responsible for the immunogenicity seen in patients. The initial aim of this study was to develop solid-phase enzyme-linked immunosorbent assays (ELISA) and an in vitro cell-based method to accurately detect ADA and estimate its impact on the preclinical in vivo efficacy outcomes of two novel, nonimmunoglobulin VNAR fusion anti-hTNF-α biologics (Quad-X™ and D1-NDure™-C4) and Humira®, a brand of adalimumab. Serum drug levels and the presence of ADA were determined in a transgenic mouse model of polyarthritis (Tg197) when Quad-X™ and Humira® were dosed at 1 mg/kg and D1-NDure™-C4 was dosed at 30 mg/kg. The serum levels of the Quad-X™ and D1-NDure™-C4 modalities were consistently high and comparable across all mice within the same treatment groups. In 1 mg/kg and 3 mg/kg Quad-X™- and 30 mg/kg D1-NDure™-C4-treated mice, an average trough drug serum concentration of 8 μg/mL, 50 μg/mL, and 350 μg/mL, respectively, were estimated. In stark contrast, Humira® trough serum concentrations in the 1 mg/kg treatment group ranged from <0.008 μg/mL to 4 μg/mL with trace levels detected in 7 of the 8 animals treated. Trough serum Humira® and Quad-X™ concentrations in 3 mg/kg treatment samples were comparable; however, the functionality of the detected Humira® serum was significantly compromised due to neutralising ADA. The impact of ADA went beyond the simple and rapid clearance of Humira®, as 7/8 serum samples also showed no detectable capacity to neutralise hTNF-α-mediated cytotoxicity in a murine fibrosarcoma (L929) cell assay. The neutralisation capacity of all the VNAR constructs remained unchanged at the end of the experimental period (10 weeks). The data presented in this manuscript goes some way to explain the exciting outcomes of the previously published preclinical in vivo efficacy data, which showed complete control of disease at Quad-X™ concentrations of 0.5 mg/kg, equivalent to 10x the in vivo potency of Humira®. This independent corroboration also validates the robustness and reliability of the assay techniques reported in this current manuscript, and while it comes with the caveat of a mouse study, it does appear to suggest that these particular VNAR constructs, at least, are of low inherent immunogenicity.

scholarly journals Indirect cholinergic activation slows down pancreatic cancer growth and tumor-associated inflammation

2020 ◽  
Vol 39 (1) ◽  
Author(s):  
Paulo L. Pfitzinger ◽  
Laura Fangmann ◽  
Kun Wang ◽  
Elke Demir ◽  
Engin Gürlevik ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Mouse Model ◽  
Tumor Stage ◽  
Ache Inhibitors ◽  
Impact On Survival ◽  
The Impact ◽  
Ache Expression ◽  
Cholinergic Activation

Abstract Background Nerve-cancer interactions are increasingly recognized to be of paramount importance for the emergence and progression of pancreatic cancer (PCa). Here, we investigated the role of indirect cholinergic activation on PCa progression through inhibition of acetylcholinesterase (AChE) via clinically available AChE-inhibitors, i.e. physostigmine and pyridostigmine. Methods We applied immunohistochemistry, immunoblotting, MTT-viability, invasion, flow-cytometric-cell-cycle-assays, phospho-kinase arrays, multiplex ELISA and xenografted mice to assess the impact of AChE inhibition on PCa cell growth and invasiveness, and tumor-associated inflammation. Survival analyses were performed in a novel genetically-induced, surgically-resectable mouse model of PCa under adjuvant treatment with gemcitabine+/−physostigmine/pyridostigmine (n = 30 mice). Human PCa specimens (n = 39) were analyzed for the impact of cancer AChE expression on tumor stage and survival. Results We discovered a strong expression of AChE in cancer cells of human PCa specimens. Inhibition of this cancer-cell-intrinsic AChE via pyridostigmine and physostigmine, or administration of acetylcholine (ACh), diminished PCa cell viability and invasion in vitro and in vivo via suppression of pERK signaling, and reduced tumor-associated macrophage (TAM) infiltration and serum pro-inflammatory cytokine levels. In the novel genetically-induced, surgically-resectable PCa mouse model, adjuvant co-therapy with AChE blockers had no impact on survival. Accordingly, survival of resected PCa patients did not differ based on tumor AChE expression levels. Patients with higher-stage PCa also exhibited loss of the ACh-synthesizing enzyme, choline-acetyltransferase (ChAT), in their nerves. Conclusion For future clinical trials of PCa, direct cholinergic stimulation of the muscarinic signaling, rather than indirect activation via AChE blockade, may be a more effective strategy.

scholarly journals CUX1—Transcriptional Master Regulator of Tumor Progression in Pancreatic Neuroendocrine Tumors

Cancers ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 1957
Author(s):  
Sebastian Krug ◽  
Julia Weissbach ◽  
Annika Blank ◽  
Aurel Perren ◽  
Johannes Haybaeck ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Mouse Model ◽  
Neuroendocrine Tumours ◽  
Tumour Progression ◽  
Free Survival ◽  
Treatment Naïve ◽  
Pancreatic Neuroendocrine Tumours ◽  
The Impact

Recently, we identified the homeodomain transcription factor Cut homeobox 1 (CUX1) as mediator of tumour de-differentiation and metastatic behaviour in human insulinoma patients. In insulinomas, CUX1 enhanced tumour progression by stimulating proliferation and angiogenesis in vitro and in vivo. In patients with non-functional pancreatic neuroendocrine tumours (PanNET), however, the impact of CUX1 remains to be elucidated. Here, we analysed CUX1 expression in two large independent cohorts (n = 43 and n = 141 tissues) of non-functional treatment-naïve and pre-treated PanNET patients, as well as in the RIP1Tag2 mouse model of pancreatic neuroendocrine tumours. To further assess the functional role of CUX1, expression profiling of DNA damage-, proliferation- and apoptosis-associated genes was performed in CUX1-overexpressing Bon-1 cells. Validation of differentially regulated genes was performed in Bon-1 and QGP1 cells with knock-down and overexpression strategies. CUX1 expression assessed by a predefined immunoreactivity score (IRS) was significantly associated with shorter progression-free survival (PFS) of pre-treated PanNET patients (23 vs. 8 months; p = 0.005). In treatment-naïve patients, CUX1 was negatively correlated with grading and recurrence-free survival (mRFS of 39 versus 8 months; p = 0.022). In both groups, high CUX1 levels indicated a metastatic phenotype. Functionally, CUX1 upregulated expression of caspases and death associated protein kinase 1 (DAPK1), known as mediators of tumour progression and resistance to cytotoxic drugs. This was also confirmed in both cell lines and human tissues. In the RIP1Tag2 mouse model, CUX1 expression was associated with advanced tumour stage and resistance to apoptosis. In summary, we identified the transcription factor CUX1 as mediator of tumour progression in non-functional PanNET in vitro and in vivo, indicating that the CUX1-dependent signalling network is a promising target for future therapeutic intervention.

scholarly journals Galectin-3 levels are elevated following nintedanib treatment

2020 ◽  
Vol 11 ◽  
pp. 204062232096841
Author(s):  
Gali Epstein Shochet ◽  
Alon Pomerantz ◽  
David Shitrit ◽  
Becky Bardenstein-Wald ◽  
Kjetil Ask ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Mouse Model ◽  
Quantitative Polymerase Chain Reaction ◽  
Lung Fibroblasts ◽  
Mrna Levels ◽  
Serum Samples ◽  
In Vivo Models ◽  
Galectin 3 ◽  
The Impact

Background and Aims: Idiopathic pulmonary fibrosis (IPF) is a common and severe form of pulmonary fibrosis. Nintedanib, a triple angiokinase inhibitor, is approved for treating IPF. Galectin 3 (Gal-3) activates a variety of profibrotic processes. Currently, the Gal-3 inhibitor TD139 is being tested in phase II clinical trials. Since this treatment is given ‘on top’ of nintedanib, it is important to estimate its effect on Gal-3 levels. Therefore, we evaluated the impact of nintedanib on Gal-3 expression using both in vitro and in vivo models, in addition to serum samples from patients with IPF. Methods: Gal-3 levels were evaluated in IPF and control tissue samples, primary human lung fibroblasts (HLFs) following nintedanib treatment (10–100 nM, quantitative polymerase chain reaction), and in a silica-induced fibrosis mouse model with/without nintedanib (0.021–0.21 mg/kg) by immunohistochemistry. In addition, Gal-3 levels were analyzed in serum samples from 41 patients with interstitial lung disease patients with/without nintedanib treatment by ELISA. Results: Nintedanib addition to HLFs resulted in significant elevations in Gal-3, phospho-signal transducer and activator of transcription 3 (pSTAT3), as well as IL-8 mRNA levels ( p < 0.05). Gal-3 expression was higher in samples from IPF patients compared with non-IPF controls at the protein and mRNA levels ( p < 0.05). In the in vivo mouse model, Gal-3 levels were increased following fibrosis induction and even further increased with the addition of nintedanib, mostly in macrophages ( p < 0.05). Patients receiving nintedanib presented with higher Gal-3 serum levels compared with those who did not receive nintedanib ( p < 0.05). Conclusion: Nintedanib elevates Gal-3 levels in both experimental models, along with patient samples. These findings highlight the possibility of using combined inhibition therapy for patients with IPF.

scholarly journals Indirect cholinergic activation slows down pancreatic cancer growth and tumor-associated inflammation

2020 ◽  
Author(s):  
Paulo L. Pfitzinger ◽  
Laura Fangmann ◽  
Kun Wang ◽  
Elke Demir ◽  
Engin Gürlevik ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Mouse Model ◽  
Tumor Stage ◽  
Ache Inhibitors ◽  
Impact On Survival ◽  
The Impact ◽  
Ache Expression ◽  
Cholinergic Activation

Abstract Background: Nerve-cancer interactions are increasingly recognized to be of paramount importance for the emergence and progression of pancreatic cancer (PCa). Here, we investigated the role of indirect cholinergic activation on PCa progression through inhibition of acetylcholinesterase (AChE) via clinically available AChE-inhibitors, i.e. physostigmine and pyridostigmine.Methods: We applied immunohistochemistry, immunoblotting, MTT-viability, invasion, flow-cytometric-cell-cycle-assays, phospho-kinase arrays, multiplex ELISA and xenografted mice to assess the impact of AChE inhibition on PCa cell growth and invasiveness, and tumor-associated inflammation. Survival analyses were performed in a novel genetically-induced, surgically-resectable mouse model of PCa under adjuvant treatment with gemcitabine+/-physostigmine/pyridostigmine (n=30 mice). Human PCa specimens (n=39) were analyzed for the impact of cancer AChE expression on tumor stage and survival.Results: We discovered a strong expression of AChE in cancer cells of human PCa specimens. Inhibition of this cancer-cell-intrinsic AChE via pyridostigmine and physostigmine, or administration of acetylcholine (ACh), diminished PCa cell viability and invasion in vitro and in vivo via suppression of pERK signaling, and reduced tumor-associated macrophage (TAM) infiltration and serum pro-inflammatory cytokine levels. In the novel genetically-induced, surgically-resectable PCa mouse model, adjuvant co-therapy with AChE blockers had no impact on survival. Accordingly, survival of resected PCa patients did not differ based on tumor AChE expression levels. Patients with higher-stage PCa also exhibited loss of the ACh-synthesizing enzyme, choline-acetyltransferase (ChAT), in their nerves.Conclusion: For future clinical trials of PCa, direct cholinergic stimulation of the muscarinic signaling, rather than indirect activation via AChE blockade, may be a more effective strategy.

scholarly journals Chronic sodium bromide relieves autistic-like deficits in the Oprm1 mouse model of autism and modulates the activity of serotonin and dopamine receptors in vitro

2020 ◽  
Author(s):  
Cécile Derieux ◽  
Sébastien Roux ◽  
Thierry Plouvier ◽  
Audrey Léauté ◽  
Agathe Brugoux ◽  
...  
Keyword(s):  
Mouse Model ◽  
Dopamine Receptors ◽  
Therapeutic Potential ◽  
Autism Spectrum ◽  
Chloride Ions ◽  
Sodium Bromide ◽  
Bromide Ion ◽  
The Impact

Chronic sodium bromide relieves autistic-like deficits in the Oprm1 mouse model of autism and modulates the activity of serotonin and dopamine receptors in vitro C. DERIEUX 1 , S. ROUX 1 , A. LEAUTE 1 , T. PLOUVIER 2 , J.A.J. BECKER 1 , J. LE MERRER 1 1 Déficits de Récompense, GPCRs et Sociabilité, Physiologie de la Reproduction et des Comportements, INRA UMR0085, CNRS UMR7247, Université de Tours, Inserm ; 37380 Nouzilly, France 2 Térali Innov, 37230 Fondettes, France Corresponding author : [email protected] Autism spectrum disorders (ASD) are complex neurodevelopmental diseases whose diagnosis lies on the detection of impaired social skills together with restricted and repetitive behavior and interests (DSM-5). Although the etiology of ASD remains mostly unknown, impaired excitation/inhibition ratio appears as a common mechanistic feature. Bromide ion is known to reduce hyperexcitability, possibly by competing with chloride ions at channels and transporters and may thus have therapeutic potential in ASD. Aims : We evaluated the therapeutic potential of bromide ion in the Oprm1 -/- mouse model of ASD and the molecular mechanisms involved in bromide treatment, notably effects on GPCRs. Methods : In vivo , we first assessed the effect of chronically administered sodium bromide on autistic-like behavioral deficits and performed RT-qPCR on brain structures known to be involved in ASD. In vitro , we evaluated the impact of bromide ion on G-protein mediated signaling of serotonin and dopamine receptors. Results : In vivo , sodium bromide (30 to 500 mg/Kg) dose-dependently improved social interaction and preference, reduced stereotypies and decreased anxiety. Bromide also impacts the expression of genes coding for some GPCRs, chloride transporters and GABA A subunits. In vitro , bromide behaves as a positive allosteric modulator of 5-HT 6 , 5-HT 7 and D1 receptors but not 5-HT 4 and D2 receptors. Conclusions : The beneficial effects of bromide administration in a genetic murine model of ASD and its impact on both gene expression and GPCR pharmacology predicts high translational potential in patients with autism, despite high heterogeneity in etiology and symptoms.

scholarly journals Indirect cholinergic activation slows down pancreatic cancer growth and tumor-associated inflammation

2020 ◽  
Author(s):  
Paulo L. Pfitzinger ◽  
Laura Fangmann ◽  
Kun Wang ◽  
Elke Demir ◽  
Engin Gürlevik ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Mouse Model ◽  
Tumor Stage ◽  
Ache Inhibitors ◽  
Impact On Survival ◽  
The Impact ◽  
Ache Expression ◽  
Cholinergic Activation

Abstract Background Nerve-cancer interactions are increasingly recognized to be of paramount importance for the emergence and progression of pancreatic cancer (PCa). Here, we investigated the role of indirect cholinergic activation on PCa progression through inhibition of acetylcholinesterase (AChE) via clinically available AChE-inhibitors, i.e. physostigmine and pyridostigmine. Methods We applied immunohistochemistry, immunoblotting, MTT-viability, invasion, flow-cytometric-cell-cycle-assays, phospho-kinase arrays, multiplex ELISA and xenografted mice to assess the impact of AChE inhibition on PCa cell growth and invasiveness, and tumor-associated inflammation. Survival analyses were performed in a novel genetically-induced, surgically-resectable mouse model of PCa under adjuvant treatment with gemcitabine+/-physostigmine/pyridostigmine (n = 30 mice). Human PCa specimens (n = 39) were analyzed for the impact of cancer AChE expression on tumor stage and survival. Results We discovered a strong expression of AChE in cancer cells of human PCa specimens. Inhibition of this cancer-cell-intrinsic AChE via pyridostigmine and physostigmine, or administration of acetylcholine (ACh), diminished PCa cell viability and invasion in vitro and in vivo via suppression of pERK signaling, and reduced tumor-associated macrophage (TAM) infiltration and serum pro-inflammatory cytokine levels. In the novel genetically-induced, surgically-resectable PCa mouse model, adjuvant co-therapy with AChE blockers had no impact on survival. Accordingly, survival of resected PCa patients did not differ based on tumor AChE expression levels. Patients with higher-stage PCa also exhibited loss of the ACh-synthesizing enzyme, choline-acetyltransferase (ChAT), in their nerves. Conclusion For future clinical trials of PCa, direct cholinergic stimulation of the muscarinic signaling, rather than indirect activation via AChE blockade, may be a more effective strategy.

scholarly journals MiR-30a-5p regulates GLT-1 function via a PKCα-mediated ubiquitin degradation pathway in a mouse model of Parkinson’s disease

2020 ◽  
Author(s):  
Xingjun Meng ◽  
Jianping Zhong ◽  
Chong Zeng ◽  
Ken Kin Lam Yung ◽  
Xiuping Zhang ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Mouse Model ◽  
Cell Model ◽  
Glutamate Transporters ◽  
Acid Uptake ◽  
Dependent Manner ◽  
The Impact

Abstract Background:Glutamate excitotoxicity caused by dysfunctional glutamate transporters plays an important role in the pathogenesis of Parkinson’s disease (PD); however, the mechanisms that underlie the regulation of glutamate transporters in PD are still not fully elucidated. MicroRNAs have been reported to play key roles in regulating the translation of glutamate-transporter mRNA. Methods: We established model of PD 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice in vivo and 1-methyl-4-phenylpyridinium (MPP+) treated astrocyte in vitro. Stereotaxic injection of shRNA in mouse, and miRNA inhibitor/mimic, or antagonist/agonist treated the cell model, Behavioral experiments, glutamic acid uptake, transport activity of synaptosomes, underlying mechanisms and the impact on neuronal survival were assessed.Results We demonstrated that short-hairpin RNA-mediated knockdown of miR-30a-5p ameliorated motor deficits and pathological changes like astrogliosis and reactive microgliosis in a mouse model of PD. Western blotting and immunofluorescent labeling revealed that miR-30a-5p suppressed the expression and function of GLT-1 in MPTP-treated mice and specifically in astrocytes treated with (cell model of PD). Conclusion Both in vitro and in vivo, we found that miR-30a-5p knockdown promoted glutamate uptake and increased GLT-1 expression by hindering GLT-1 ubiquitination and subsequent degradation in a PKCα-dependent manner. Therefore, miR-30a-5p represents a potential therapeutic target for the treatment of PD.

The HSP90 Inhibitor 17-DMAG Targets NF-κB to Induce Apoptosis in CLL and Prolongs Survival in a CLL Mouse Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 732-732
Author(s):  
Erin K Hertlein ◽  
Thomas S Lin ◽  
Wagner J Amy ◽  
Towns H William ◽  
Virginia M Goettl ◽  
...  
Keyword(s):  
Mouse Model ◽  
Disease Progression ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Therapeutic Option ◽  
Hsp90 Inhibitor ◽  
Client Proteins ◽  
The Impact

Abstract Abstract 732 One attractive therapeutic target currently being explored in CLL is HSP90, a chaperone which stabilizes various client proteins (AKT, Raf, ZAP-70) which are important for survival of CLL cells. Interfering with HSP90 protein binding to these client proteins leads to their rapid degradation. Our group and others have demonstrated that 17-allylamino-17-demethoxy-geldanamycin (17-AAG) depletes only select chaperone proteins and promotes modest cytotoxicity in CLL patient cells. 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG) is a novel HSP90 inhibitor with improved solubility, bioavailability and cytotoxicity in cancer cell lines as compared to 17-AAG. We demonstrate that 17-DMAG more potently induces caspase-dependent apoptosis of primary CLL cells compared to 17-AAG. This enhanced cytotoxicity is tumor cell selective as it spares normal T-cells and NK-cells, and only modestly affects normal B cells. In addition to the broad range of Hsp90 client proteins which are regulated by 17-AAG (AKT, CDK9, ZAP-70), we found that 17-DMAG was also able to deplete both IKKαa and IKKβ, the activating kinases of the NF-κB family of transcription factors. The impact of 17-DMAG on both IKKαa and β is very relevant, as there have been several studies investigating the effect of IKK inhibitors on in vitro apoptosis in CLL, however there is still no clear therapeutic option for targeting these kinases in the clinic. Most IKK inhibitors including 17-AAG are specific for the IKKβ subunit that mediates classical NF-κB signaling, leaving IKKαa and the alternative NF-κB pathway intact. This is particularly important given recent evidence by Lam et al. (Clin Can Res 2005 Jan 1;11(1):28-40) demonstrating that IKKαa is able to compensate for the loss of IKKβ in DLBCL cells lines. Therefore the ability of 17-DMAG to target both subunits of the IKK complex potentially makes it a very potent and effective NF-κB inhibitor in CLL. To validate the downstream significance of this NF-κB regulation, we show that 17-DMAG effectively reduces NF-κB nuclear localization DNA binding in CLL patient cells resulting in decreased transcript and protein levels of NF-κB targets MCL1 and BCL2, known to be major factors in CLL cell survival and drug resistance. The decrease in MCL1 and BCL2 following 17-DMAG treatment was not prevented by treatment with the caspase inhibitor zVAD-fmk even though viability was rescued, suggesting that the decrease in these survival proteins precedes cell death and is not simply a consequence of the apoptotic process. Finally, we determined the in vivo significance of 17-DMAG treatment using a TCL1-SCID transplant model. We found that NF-κB targets genes (A20, BCL2, cIAP, MCL1 and XIAP) were decreased in vivo following treatment with 17-DMAG. In addition to typical genes which are activated by NF-κB, we also determined the in vivo effect of 17-DMAG on NF-κB mediated transcriptional repression. Our group has recently determined that the transcription factor FOXD3 is transcriptionally silenced by an NF-κB repressor complex very early in the disease progression of the TCL1 mouse model, an event which facilitates subsequent global DNA methylation and gene silencing in CLL. We found that treatment with 17-DMAG leads to re-expression of FOXD3 suggesting that 17-DMAG is an effective therapeutic tool to target NF-κB mediated gene repression as well as NF-κB mediated expression of survival genes. Furthermore, 17-DMAG treatment significantly prolonged the survival of these mice (75 days vs. 66 days, p=0.027, n=10/group). Together, our data demonstrate that the HSP90 inhibitor 17-DMAG represents a novel multi-subunit IKK inhibitor leading to a decrease in anti-apoptosis genes relevant to CLL survival while increasing the expression of genes silenced during CLL disease progression. Given its oral formulation, which allows administration of 17-DMAG by continuous dosing and uninterrupted inhibition of HSP90, initiation of phase II clinical trials in CLL that include detailed pharmacodynamic studies monitoring NF-κB target genes are indicated. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Chronic sodium bromide relieves autistic-like deficits in the Oprm1 mouse model of autism and modulates the activity of serotonin and dopamine receptors in vitro

2020 ◽  
Author(s):  
Cécile Derieux ◽  
Sébastien Roux ◽  
Thierry Plouvier ◽  
Audrey Léauté ◽  
Agathe Brugoux ◽  
...  
Keyword(s):  
Mouse Model ◽  
Dopamine Receptors ◽  
Therapeutic Potential ◽  
Autism Spectrum ◽  
Chloride Ions ◽  
Sodium Bromide ◽  
Bromide Ion ◽  
The Impact

Chronic sodium bromide relieves autistic-like deficits in the Oprm1 mouse model of autism and modulates the activity of serotonin and dopamine receptors in vitro C. DERIEUX 1 , S. ROUX 1 , A. LEAUTE 1 , T. PLOUVIER 2 , J.A.J. BECKER 1 , J. LE MERRER 1 1 Déficits de Récompense, GPCRs et Sociabilité, Physiologie de la Reproduction et des Comportements, INRA UMR0085, CNRS UMR7247, Université de Tours, Inserm ; 37380 Nouzilly, France 2 Térali Innov, 37230 Fondettes, France Corresponding author : [email protected] Autism spectrum disorders (ASD) are complex neurodevelopmental diseases whose diagnosis lies on the detection of impaired social skills together with restricted and repetitive behavior and interests (DSM-5). Although the etiology of ASD remains mostly unknown, impaired excitation/inhibition ratio appears as a common mechanistic feature. Bromide ion is known to reduce hyperexcitability, possibly by competing with chloride ions at channels and transporters and may thus have therapeutic potential in ASD. Aims : We evaluated the therapeutic potential of bromide ion in the Oprm1 -/- mouse model of ASD and the molecular mechanisms involved in bromide treatment, notably effects on GPCRs. Methods : In vivo , we first assessed the effect of chronically administered sodium bromide on autistic-like behavioral deficits and performed RT-qPCR on brain structures known to be involved in ASD. In vitro , we evaluated the impact of bromide ion on G-protein mediated signaling of serotonin and dopamine receptors. Results : In vivo , sodium bromide (30 to 500 mg/Kg) dose-dependently improved social interaction and preference, reduced stereotypies and decreased anxiety. Bromide also impacts the expression of genes coding for some GPCRs, chloride transporters and GABA A subunits. In vitro , bromide behaves as a positive allosteric modulator of 5-HT 6 , 5-HT 7 and D1 receptors but not 5-HT 4 and D2 receptors. Conclusions : The beneficial effects of bromide administration in a genetic murine model of ASD and its impact on both gene expression and GPCR pharmacology predicts high translational potential in patients with autism, despite high heterogeneity in etiology and symptoms.

68Ga-labeled HBED-CC Variant of uPAR Targeting Peptide AE105 Compared with 68Ga-NODAGA-AE105

2019 ◽  
Vol 18 (9) ◽  
pp. 1289-1294 ◽  
Author(s):  
Kusum Vats ◽  
Rohit Sharma ◽  
Haladhar D. Sarma ◽  
Drishty Satpati ◽  
Ashutosh Dash
Keyword(s):  
Solid Phase ◽  
Evaluation Studies ◽  
Radiochemical Yield ◽  
In Vivo Evaluation ◽  
Peptide Conjugates ◽  
Biodistribution Studies ◽  
Target Organs ◽  
U87mg Tumor

Aims: The urokinase Plasminogen Activator Receptors (uPAR) over-expressed on tumor cells and their invasive microenvironment are clinically significant molecular targets for cancer research. uPARexpressing cancerous lesions can be suitably identified and their progression can be monitored with radiolabeled uPAR targeted imaging probes. Hence this study aimed at preparing and evaluating two 68Ga-labeled AE105 peptide conjugates, 68Ga-NODAGA-AE105 and 68Ga-HBED-CC-AE105 as uPAR PET-probes. Method: The peptide conjugates, HBED-CC-AE105-NH2 and NODAGA-AE105-NH2 were manually synthesized by standard Fmoc solid phase strategy and subsequently radiolabeled with 68Ga eluted from a commercial 68Ge/68Ga generator. In vitro cell studies for the two radiotracers were performed with uPAR positive U87MG cells. Biodistribution studies were carried out in mouse xenografts with the subcutaneously induced U87MG tumor. Results: The two radiotracers, 68Ga-NODAGA-AE105 and 68Ga-HBED-CC-AE105 that were prepared in >95% radiochemical yield and >96% radiochemical purity, exhibited excellent in vitro stability. In vivo evaluation studies revealed higher uptake of 68Ga-HBED-CC-AE105 in U87MG tumor as compared to 68Ga-NODAGAAE105; however, increased lipophilicity of 68Ga-HBED-CC-AE105 resulted in slower clearance from blood and other non-target organs. The uPAR specificity of the two radiotracers was ascertained by significant (p<0.05) reduction in the tumor uptake with a co-injected blocking dose of unlabeled AE-105 peptide. Conclusion: Amongst the two radiotracers studied, the neutral 68Ga-NODAGA-AE105 with more hydrophilic chelator exhibited faster clearance from non-target organs. The conjugation of HBED-CC chelator (less hydrophilic) resulted in negatively charged 68Ga-HBED-CC-AE105 which was observed to have high retention in blood that decreased target to non-target ratios.

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